Fluorescence anisotropy: from single molecules to live cells.
ABSTRACT The polarization of light emitted by fluorescent probes is an easily accessible physical quantity that is related to a multitude of molecular parameters including conformation, orientation, size and the nanoscale environment conditions, such as dynamic viscosity and temperature. In analytical biochemistry and analytical chemistry applied to biological problems, fluorescence anisotropy is widely used for measuring the folding state of proteins and nucleic acids, and the affinity constant of ligands through titration experiments. The emphasis of this review is on new multi-parameter single-molecule detection schemes and their bioanalytical applications, and on the use of ensemble polarization assays to study binding and conformational dynamics of proteins and aptamers and for high-throughput discovery of small-molecule drugs.
Article: Electrons, photons, and force: quantitative single-molecule measurements from physics to biology.[show abstract] [hide abstract]
ABSTRACT: Single-molecule measurement techniques have illuminated unprecedented details of chemical behavior, including observations of the motion of a single molecule on a surface, and even the vibration of a single bond within a molecule. Such measurements are critical to our understanding of entities ranging from single atoms to the most complex protein assemblies. We provide an overview of the strikingly diverse classes of measurements that can be used to quantify single-molecule properties, including those of single macromolecules and single molecular assemblies, and discuss the quantitative insights they provide. Examples are drawn from across the single-molecule literature, ranging from ultrahigh vacuum scanning tunneling microscopy studies of adsorbate diffusion on surfaces to fluorescence studies of protein conformational changes in solution.ACS Nano 02/2011; 5(2):693-729. · 10.77 Impact Factor